Larynx cancer (LCa) is an aggressive malignancy, which is the second most common malignant neoplasm of head and neck squamous cell carcinoma. Its incidences have been reported to increase and therapeutic options mostly fail to give positive clinical response especially for the advanced LCa cases. In this study we aimed to isolate stem-like cells from freshly resected LCa tumor specimens and characterize them by quantitative real time PCR (qRT-PCR) for expression of cancer stem cell markers including SOX2, OCT4, KLF4, ABCG2, CXCR4 and CD44. Our results showed that CD133(high) cells directly isolated from freshly resected tumor specimens exhibit elevated levels of SOX2, OCT4 and KLF4, and have increased expression levels of ABCG2 and CXCR4, which were associated with resistance of tumors to regular chemotherapeutic reagents. In conclusion, this study offers a useful approach utilizing CD133 to isolate stem cells directly from fresh tissues, which gives the opportunity to develop novel therapeutic tools specifically targeting these cells through their further characterization. © 2014 Bentham Science Publishers.